The present invention relates generally to the field of malignant cell proliferation. More particularly, it provides compositions and methods to limit Bcr-Abl oncoprotein-driven malignant cell proliferation. Peptide and protein molecules are provided that inhibit various Bcr-Abl signal transduction pathways, e.g., activation of the Ras protein. Methods for reducing Philadelphia chromosome-positive cells in cell populations, including bone marrow culture, and methods of treating various leukemias are also provided.
The Philadelphia chromosome (Ph1) is associated with the bulk of chronic myelogenous leukemia (CML) patients (more than 95%), 10-25% of acute lymphocytic leukemia (ALL) patients, and about 2-3% of acute myelogenous leukemias (AML). This abnormal chromosome fuses most of the ABL gene to the 5xe2x80x2 two-thirds of the BCR gene.
A number of different kinds of evidence support the contention that Bcr-Abl oncoproteins, such as p210 and p185 BCR-ABL, are causative factors in these leukemias (Campbell et al., 1991). The malignant activity is due in large part to the Bcr-Abl protein""s highly activated protein tyrosine kinase activity and its abnormal interaction with protein substrates (Campbell et al., 1991, Arlinghaus et al., 1990). The Bcr-Abl oncoprotein p210 Bcr-Abl is associated with both CML and ALL, whereas the smaller oncoprotein, p185 BCR-ABL, is associated with ALL patients, although some CML patients also express p185 (Campbell et al., 1991).
Some reports suggest that Bcr-Abl oncoproteins, p210 and p185 BCR-ABL, function at least in part by activating the Ras pathway. The RAS gene is a proto-oncogene involved in controlling normal cell growth. When continuously activated, the Ras protein becomes a potent cancer gene product. Bcr-Abl oncoproteins have been observed by the present inventors and others to perturb normal Ras function (Pendergast et al., 1993).
The mechanism by which Bcr-Abl oncoproteins activate p21 Ras is believed to involves several factors. One event involves the autophosphorylation of the Bcr-Abl oncoprotein on tyrosine residues within the coding sequence of the first Bcr exon (Liu et al., 1993). This finding was unexpected, as it had previously been postulated that Bcr-Abl phosphorylates itself on Abl tyrosines, not Bcr tyrosine residues.
Several adaptor proteins have been implicated in Ras-activation as well. FIG. 2 lists several such adaptor proteins that contain SH2/SH3 motifs. Such domains have been observed in proteins involved in transmitting growth signals to the nucleus (Pawson et al., 1992).
Grb2 is an adaptor protein that binds to tyrosine phosphorylated receptor proteins. Bcr-Abl induced oncogenesis has also been reported to be mediated by direct interaction with the SH2 domain of Grb2 (Pendergast et al., 1993; Puil et al., 1994). Grb2 also binds mSos1, a GTP exchange factor (see FIG. 3). The latter activates Ras by forming GTP/Ras. GTP/Ras in turn activates Raf, a serine/threonine protein kinase that activates Mek. Mek is a kinase that phosphorylates and activates MAP kinase. The latter is believed to activate and/or regulate various transcription factors (i.e., c-Jun), resulting in cell growth (FIG. 4).
Another peptide that has been implicated in the malignant effects of Bcr-Abl involves Shc (Puil et al., 1994). Crkl is another adaptor molecule that forms a protein/protein interaction with Bcr-Abl (Reichman et al., 1992; Ten Hoeve et al., 1993; 1994). Still another adaptor molecule that interacts with Bcr-Abl is p120 Ras Gap (Druker et al. 1992).
Another protein/protein interaction that has been examined in relation to Bcr-Abl induced malignancy concerns the formation of tetramer structures. In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl.
Activation of the Abl transforming function is believed to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176-242) (McWhirter et al., 1993). Domain 1 of Bcr has been shown to form a homotetramer (McWhirter et al., 1993). The Bcr-Abl tetramer activates its inherent Abl tyrosine kinase activity, its actin binding function, and its cellular transformation function (McWhirter et al., 1993). Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function and the ability of Bcr-Abl to transform Rat-1 fibroblasts.
Despite the description of certain events and molecules that are believed to be involved in Bcr/Abl function and pathologies associated with the activities of its gene product, comprehensive strategies for controlling Bcr/Abl and, e.g., its activation of the Ras oncogene, have not been developed. Thus, a need continues to exist in the scientific and medical arts for approaches that target effectively and specifically inhibit Bcr/Abl. Such techniques would provide new therapies for inhibiting Philadelphia chromosome-positive cells in tissues, such as in bone marrow.
The present invention overcomes certain of the limitations of the prior art by defining specific peptide sequences from Bcr-Abl that inhibit Bcr-Abl function and activation. The peptides and compositions of the invention are thus useful in methods for inhibiting Bcr-Abl, for purging bone marrow of Philadelphia chromosome-positive cells in bone marrow samples and for treating various leukemias, including chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL) and acute myelogenous leukemias (AML).
The peptides of the present invention are those comprising a sequence based upon a segment of a Bcr-Abl amino acid sequence that includes at least one of a combination of several tyrosine residues found by the present inventors to be important in Bcr-Abl function. These are termed herein the xe2x80x9ctyrosine-containing peptidesxe2x80x9d. Generally, the compositions and methods of the invention require that at least one tyrosine-containing peptide be present.
The amino acid sequence of the first exon of p160 Bcr is given in SEQ ID NO:1. Tyrosines are present at residues 58, 70, 177, 231, 246, 276, 279, 283, 316, 328 and 360. The particularly important tyrosines in the context of the present invention are tyrosines at positions 177, 283 and 360, and also tyrosine 328.
The present invention thus provides purified peptides and polypeptides, of between about 4 and about 500 amino acids in length, that have or comprise a contiguous amino acid sequence from the Bcr-Abl protein (of SEQ ID NO:1), which sequence includes or surrounds at least one of tyrosine 177, tyrosine 283, tyrosine 360, or even tyrosine 328, which peptides or polypeptides become phosphorylated on tyrosine 177, 283, 360 and/or 328 upon contact with active Bcr-Abl.
These peptides are thus characterized as being substrates for the tyrosine phosphorylating activity of Bcr-Abl. The peptides are also characterized as being capable of effectively competing with Bcr as a substrate for Bcr-Abl, and being capable of reducing the Bcr-Abl-mediated tyrosine phosphorylation of Bcr in an intact cell that contains Bcr-Abl.
Exemplary useful peptides including tyrosine 177 are those comprising the sequence of SEQ ID NO:8 (positions 164 to 181 of SEQ ID NO:1). Exemplary useful peptides including tyrosine 283 are those comprising the sequence of SEQ ID NO:11 (positions 255 to 293 of SEQ ID NO:1). It is currently preferred that the peptide include the tyrosine in a generally central region, rather than at the extreme termini of the peptide.
Although understanding the mechanism of action of any given peptide is not necessary in order to practice the invention, it should be noted that peptides containing tyrosine 177 or tyrosine 283 likely function by inhibiting the oncogenic effects of Bcr-Abl. This is believed to be achieved by the peptides competing with other substrates, particularly Bcr, in order to become phosphorylated by Bcr-Abl. In effect, this reduces the Bcr-Abl-driven phosphorylation of other cellular targets and limits the adverse effects of Bcr-Abl.
Still further useful tyrosine-containing peptides are those containing tyrosine 360. Exemplary useful peptides of this group include those comprising the sequence of SEQ ID NO:10 (positions 353 to 364 of SEQ ID NO:1) and SEQ ID NO:22 (positions 350 to 366 of SEQ ID NO:1). It is currently preferred that the a phosphorylated serine, corresponding to Serine 354, be provided in these peptides.
The advantageous effects of peptides containing tyrosine 360 are believed to be based on the stimulation of the beneficial effects of Bcr, which neutralizes Bcr-Abl. Such Bcr neutralization is achieved, in part, by its Serine/Threonine kinase activity. Phosphorylation of Bcr tyrosine 360 and tyrosine 328 by Bcr-Abl has been shown by the inventors to reduce its Serine/Threonine phosphorylating activity. Importantly, the Ser 354 form of a tyrosine 360-containing peptide is demonstrated herein to be a direct inhibitor of the Bcr-Abl oncoprotein""s tyrosine kinase activity.
Therefore, peptides containing tyrosine 360 sequences, or even tyrosine 328 sequences, will compete with Bcr as a Bcr-Abl substrate and will reduce the levels of Bcr-Tyr-360-P, thereby facilitating the beneficial effects of Bcr. However, it will again be understood that the present invention is still useful even if this proposed mechanism of action does not prove to be entirely correct.
In terms of the tyrosine-containing peptides of the invention, peptides that include at least a 4-mer or 5-mer sequence, or preferably, that include at least a 6-mer or 7-mer sequence, that includes the tyrosine of importance are expected to provide effective molecules in the compositions for Bcr-Abl inhibition. For example, a sequence that includes at least the amino acid sequence of SEQ ID NO:24 (positions 176 to 180 of SEQ ID NO:1) may be employed. Exemplary 5-and 7-mers are represented by SEQ ID NO:25 (positions 359 to 363 of SEQ ID NO:1) and SEQ ID NO:26 (positions 279 to 285 of SEQ ID NO:1).
However, longer peptides, from about 10-12 to 15-20, to about 50, 100, 150, 200, 250, 300, 350, 400 or about 500 residues or so may also be used. An exemplary 13-mer is SEQ ID NO:27, corresponding to positions 168 to 180 of SEQ ID NO:1. A currently preferred longer peptide is that of SEQ ID NO:28.
Shorter peptides, such as SEQ ID NO:10 and SEQ ID NO:22, will generally be administered to cells or patients as a peptide or liposomal-peptide formulation. Longer peptides and polypeptides, such as SEQ ID NO:28, will generally be administered to cells or patients using gene therapy, in which a vector that expresses the peptide or polypeptide is employed.
While a given peptide alone will be useful in inhibiting Bcr-Abl, the compositions and methods of the present invention may include two or more such peptides. Where two peptides are employed, it may be preferred to use peptides with sequences including distinct tyrosine regions, preferably selected from those regions of SEQ ID NO:1 including 177, 283 and/or 360. Three distinct peptides including the foregoing various tyrosine regions may also be used to advantage.
A single peptide that itself contains sequences surrounding two of the three tyrosines at positions 177, 283 and 360 may also be used. Such peptides may contain only contiguous Bcr-Abl sequences, or may contain two contiguous stretches of Bcr-Abl sequence operatively joined by an irrelevant, preferably flexible, linker sequence.
Further, the use of a single peptide that contains a contiguous sequence that includes each of the three tyrosines at positions 177, 283 and 360 is also contemplated. An exemplary peptide containing only contiguous Bcr-Abl sequences is SEQ ID NO:28, which begins at residue 64 and ends at residue 413 of SEQ ID NO:1. Peptides may also contain longer stretches of Bcr-Abl sequences or other sequences as desired.
Other peptides of the present invention that may be used in addition to at least one tyrosine-containing peptide are those that comprise the sequence of an important binding site on Bcr-Abl for an adaptor molecule, i.e., a molecular target of the Bcr-Abl oncoprotein. These are termed the xe2x80x9cbinding site peptidesxe2x80x9d.
The binding site peptides for use in the invention are purified peptides and polypeptides, of between about 4 and about 500 amino acids in length, that have or comprise a contiguous adaptor molecule binding site sequence from the Bcr-Abl sequence, which peptides or polypeptides bind to an adaptor molecule.
The binding site peptides may also contain tyrosine residues. However, binding site peptides are characterized as binding to an adaptor molecule, such as Grb2, Shc, Crkl, Ras Gap or an N-terminal coiled-coil region of Bcr. Preferably, they are characterized as inhibiting the binding of Bcr-Abl to an adaptor molecule, and as being capable of reducing Bcr-Abl-adaptor molecule interactions in an intact cell.
The binding site peptides for use in the compositions and methods of the invention generally mimic the sites on Bcr-Abl to which key oncoproteins bind. In these embodiments, supplementary peptides are provided that bind one or more signal transduction molecules, such as Shc, Crkl, Ras Gap and/or Grb2/mSos1, thereby preventing these molecules from carrying out their growth-promoting functions.
Accordingly, the tyrosine-containing peptide compositions of the invention may further comprise one or more of the binding proteins: a purified peptide that binds to an Abl SH3 binding protein-rich region of Shc; a purified peptide that binds to a proline-rich ABl binding site on Crkl; a purified peptide that binds to an SH2 domain of p120 Ras Gap; and/or a purified peptide or protein that binds to an N-terminal coiled-coil region of Bcr.
Exemplary compositions of the invention are those that additionally comprise one or more peptides that bind an Abl SH3 binding protein-rich region of Shc. Shc binds to Grb2 and this complex has potential to activate Ras. Peptides that bind to Shc will comprises a sequence from the Abl region, not from the Bcr region.
Further binding peptides of the present invention are those that inhibit binding of Crkl to Bcr-Abl. Crkl is a 38-kDa protein that forms complexes with both Abl and Bcr/Abl and is tyrosine phosphorylated by Abl and Bcr-Abl. Peptides that mimic the proline-rich Abl binding site on CRKL are thus also components of some embodiments of the present invention.
Still further Bcr/Abl peptides included in these embodiments are those that interact with p120 Ras Gap. Peptides of this nature are described more particularly as peptides that bind an SH2 domain of p120 Ras Gap. These peptides involve tyrosine 279 and a tyrosine outside of the first exon of Bcr. Exemplary useful peptides are those comprising the sequence of SEQ ID NO:11 (positions 255 to 293 of SEQ ID NO:1) and SEQ ID NO:12.
In further embodiments, the compositions of the invention will comprise one or more peptides or proteins that bind an N-terminal coiled-coil region of Bcr. These peptides and proteins are exemplified by peptides that comprise a sequence corresponding to SEQ ID NO:2 (positions 1 to 63 of SEQ ID NO:1); SEQ ID NO:3 (positions 1 to 71 of SEQ ID NO:1); SEQ ID NO:4 (positions 28 to 58 of SEQ ID NO:1); a sequence corresponding to SEQ ID NO:5 (positions 1 to 159 of SEQ ID NO:1); a sequence corresponding to SEQ ID NO:6 (positions 1 to 221 of SEQ ID NO:1); or a sequence corresponding to SEQ ID NO:7 (positions 1 to 413 of SEQ ID NO:1).
The present inventors observed that the Bcr protein contains the consensus binding site Y*VNV (SEQ ID NO:13) for Grb2 that enables the Bcr-Abl oncoprotein to form a complex with Grb2. Tyrosine phosphorylation of Bcr sequences at tyrosine 177 causes Grb2/mSos1 to bind membrane-bound Bcr-Abl and to activate p21 Ras (FIG. 5).
Peptides comprising the Y*VNV consensus binding site (SEQ ID NO:13; residues 177 to 180 of SEQ ID NO:1) will thus interfere with Grb2 binding to Bcr-Abl block Bcr-Abl induced malignant effects, particularly when used in combination with one or more of the Shc, Crkl, SH2 or p120 Ras Gap binding sequences. The Bcr peptide GHGQPGADAEKPFp.Y177VNVE, SEQ ID NO:8 (residues 164-181 of SEQ ID NO:1), also strongly binds to the SH2 binding site on Grb2 and may be used in the compositions of the invention.
The binding site peptides of the invention may thus be any length from between about 4 amino acids to about 500 amino acids or so, so long as the peptide is of sufficient length to include an effective binding site, as described herein.
Any of the peptide compositions of the present invention may further include a pharmaceutically acceptable carrier, such as Ringers solution, saline, and the like. Such carriers are known to those of ordinary skill in the pharmaceutical arts.
Another embodiment of the invention provides compositions comprising one or more of the above tyrosine-containing peptides in association with a liposomal formulation. The peptides may be encapsulated within the liposome or simply maintained in functional association with the liposome.
The foregoing peptide compositions may be used in enriching for Philadelphia chromosome-negative cells in a mixture of cells containing Philadelphia chromosome-positive cells.
Further provided by the present invention is a first expression vector, such as a plasmid, adenovirus or retrovirus, that contains a first DNA sequence or sequences that encodes and expresses at least one of the tyrosine-containing peptides of the invention. DNA sequences that encodes the amino acid sequence of SEQ ID NO:1 are known to those of skill in the art and are further described herein. The identification of a particular coding region that encodes one or more tyrosine-containing peptides will be straightforward to one of skill in the art.
The first expression vector may further comprise a second DNA sequence or sequences that encodes and expresses at least one of: a peptide that binds an Abl SH3 binding protein-rich region of Shc; a peptide that binds a proline-rich Abl binding site on Crkl; a peptide that binds an SH2 domain of p120 Ras Gap; a peptide or protein that binds an N-terminal coiled-coil region of Bcr; and/or a peptide that binds an SH2 binding site on Grb2.
Equally, a second expression vector, such as a plasmid, adenovirus or retrovirus, may be provided for use with the first expression vector described above. The second expression vector will generally be a plasmid, adenovirus or retrovirus that contains a DNA sequence or sequences that encodes and expresses at least one of: a peptide that binds an Abl SH3 binding protein-rich region of Shc; a peptide that binds a proline-rich Abl binding site on Crkl; a peptide that binds an SH2 domain of p120 Ras Gap; a peptide or protein that binds an N-terminal coiled-coil region of Bcr; and/or a peptide that binds an SH2 binding site on Grb2.
In certain embodiments, an amphotropic retrovirus, defective in replication but capable of infecting bone marrow cells from animals or patients, is provided that expresses one or more of the peptides of the present invention, either singly or as part of a fused polypeptide. AAV, adenoviral and plasmid vectors associated with liposomes are also provided.
Methods of the invention provide for the inhibition, killing or effective reversal of phenotype of Philadelphia chromosome-positive cells. The methods generally comprise contacting a Philadelphia chromosome-positive cell, or a population of cells that includes Philadelphia chromosome-positive cells, with a composition that includes or encodes a biologically effective amount of any one of, or a combination of, any of the tyrosine-containing peptide compositions described herein. The compositions are maintained in contact with the cells for a period of time effective to result in inhibition or killing of the Philadelphia chromosome-positive cells.
It will be understood that the methods may be achieved by contacting one or more Philadelphia chromosome-positive cells with an effective amount of one or more tyrosine-containing peptides themselves. Equally, the cells may be contacted with one or more expression vectors, including viral vectors, that encode and express one or more such tyrosine-containing peptides.
The compositions for use in such methods will generally include or encode at least one peptide having or comprising a sequence that includes at least one of the tyrosine residues 177, 283 or 360 from SEQ ID NO:1. In certain embodiments, the composition will further include or encode: a peptide that binds an Abl SH3 binding protein-rich region of Shc; a peptide that binds a proline-rich Abl binding site on Crkl; a peptide that binds an SH2 domain of p120 Ras Gap; a peptide or protein that binds an N-terminal coiled-coil region of Bcr; and/or a peptide that binds an SH2 binding site on Grb2.
In the methods for inhibiting, killing or reversing the phenotype of Philadelphia chromosome-positive cells, the cells or populations of cells may be contacted either in vivo or in vitro. The methods thus encompass both in vivo treatment and in vitrotex vivo protocols. Both in vivo and in vitro, the cells may be contacted with a composition of peptides, a composition of liposomally-associated peptides and/or with a composition comprising an expression vector or virus that encodes and expresses the peptides. The compositions will generally be pharmaceutically acceptable.
The invention thus further provides methods for enriching Philadelphia chromosome-negative cells in a mixture of cells containing Philadelphia chromosome-positive cells. The methods generally comprise contacting, for en effective period of time, a mixture of cells, such as a bone marrow sample, that contains, or is suspected of containing, Philadelphia chromosome-positive cells with a composition that includes or encodes a Bcr-Abl-inhibiting amount of any one of, or a combination of, any of the tyrosine-containing peptide compositions of the present invention. In certain embodiments, the bone marrow will be obtained from a patient having CML, AML or ALL.
The invention particularly contemplates that the bone marrow sample treated with the peptides of the invention will be re-administered to the patient from whom it was obtained. The invention thus provides methods for ex vivo treatment and bone marrow purging prior to autologous bone marrow transplants. The treated bone marrow samples enhance the immunocompetency of the transplant recipient.
In the methods for enriching Philadelphia chromosome-negative cells in a mixture of cells containing Philadelphia chromosome-positive cells, the Philadelphia chromosome-negative cells are generally enriched relative to numbers naturally occurring in a sample containing Philadelphia chromosome positive cells. An example is enriching for Philadelphia chromosome-negative cells relative to numbers naturally occurring in a bone marrow sample from a patient having CML, AML or ALL.
In still further embodiments, the invention provides methods for purging a bone marrow sample of Philadelphia chromosome-positive cells. The methods generally comprise exposing, for an effective period of time, a bone marrow sample that contains Philadelphia chromosome-positive cells to a composition that includes or encodes any one of, or a combination of, any of the tyrosine-containing peptide compositions of the present invention in an amount effective to reduce the numbers of Philadelphia chromosome-positive cells in the bone marrow sample.
Yet still further embodiments of the invention provide methods of treating a patient having or suspected of having a Philadelphia chromosome-positive leukemia, comprising treating a bone marrow sample of the patient with a composition including or encoding at least one of the tyrosine-containing peptides of the invention in an amount effective to prepare an essentially leukemia cell-free autologous bone marrow sample (i.e., using a leukemia cell-cytotoxic amount) and administering the treated sample to the patient.
The treatment methods also comprise obtaining a bone marrow sample from the patient, contacting the bone marrow sample ex vivo with a composition that includes or encodes any one or more of the tyrosine-containing peptides of the invention in a therapeutic amount and for a period of time effective to purge Philadelphia chromosome-positive cells from the bone marrow sample and re-administering the purged bone marrow sample to the patient.
For the purpose of this invention, an autologous bone marrow sample is defined as a sample of bone marrow from a patient intended for re-administration to the patient after treatment outside the body.
A defined method for treating leukemia in a patient according to the present invention comprises: administering to a patient with leukemia a chemotherapeutic regimen sufficient to generate at least some cytogenetic remission in the patient; obtaining a bone marrow sample from the patient in remission; exposing the bone marrow sample to a Philadelphia chromosome-positive cell cytotoxic concentration of tyrosine-containing peptides to provide an essentially Philadelphia chromosome-positive cell free bone marrow sample; and reintroducing the essentially Philadelphia chromosome-positive cell free bone marrow sample into the patient, wherein the reintroduction replaces Philadelphia chromosome-positive marrow cells with normal hematopoietic progenitor cells.
The approaches of the present invention provide an improvement over current strategies, e.g., anti-sense Bcr-Abl approaches, in that the activity of Bcr-Abl is inhibited while at the same time neutralizing more than one of the principal targets of Bcr-Abl oncoproteins.
The present invention also has practical uses in that the peptides mat be used as molecular weight markers, protein stain standards and as standards for radioiodination.